Certain kinds of peptides such as γ-glutamylvalylglycine (L-γ-glutamyl-L-valyl-glycine, henceforth also referred to as “γ-Glu-Val-Gly”) have a calcium sensing receptor agonist activity (Patent document 1). Such peptides having a calcium receptor agonist activity are known to be able to impart “kokumi” to foods and drinks (Patent document 2), improve tastes of low fat foods, especially fat-like thickness and smoothness (Patent document 3), improve feeling of body of sweet taste substances, and improve bitterness peculiar to sweet taste substances (Patent document 4).
Moreover, such peptides as mentioned above are known to have a prophylactic or curative effect on diarrhea (Patent document 5) and diabetes (Patent document 6), and a bicarbonate secretion promoting effect in the alimentary tract (Patent document 7).
As methods for producing γ-glutamyl tripeptides, chemical synthesis methods and enzymatic methods are generally known. As one of the chemical synthesis methods, a method of selectively obtaining a γ-glutamyl tripeptide from a dipeptide by using N-protected glutamic anhydride is known (Patent document 8). As one of the enzymatic methods, there is known a method of using glutamate-cysteine ligase and glutathione synthetase is known (Patent documents 9 and 10). As another enzymatic method, there is also known a method of γ-glutamylating Val-Gly by using γ-glutamyltransferase to generate γ-Glu-Val-Gly (Patent document 11).
Glutamate-cysteine ligase (GSHA) is an enzyme having an activity for catalyzing the reaction of generating γ-Glu-Cys, ADP, and phosphate using Glu, Cys, and ATP as substrates (EC 6.3.2.2). GSHA usually requires divalent metal ions such as Mg2+ and Mn2+ for the enzymatic reaction.
GSHA of Escherichia coli generates γ-glutamyl peptides using Glu, various kinds of amino acids, and ATP as substrates in the presence of Mg2+ or Mn2+, and it is known that type of the metal ion serving as a cofactor affects the substrate specificity thereof (Non-patent document 1). Specifically, it has been reported that when Mg2+ is used as the cofactor, Vmax is 251 mol/mg/hr and Km is 17.6 mM as for the γ-Glu-Gly generation activity, whereas Vmax is 59 mol/mg/hr and Km is 27.1 mM as for the γ-Glu-Val generation activity. That is, if the activities are compared by using Vmax/Km as index of the activities, the ratio of γ-Glu-Val generation activity to the γ-Glu-Gly generation activity in the case of using Mg2+ as the cofactor can be calculated to be 0.15. Further, it has been demonstrated that when Mn2+ is used as the cofactor, Vmax is 39 mol/mg/hr and Km is 1.7 mM as for the γ-Glu-Gly generation activity, whereas Vmax is 95 mol/mg/hr and Km is 21 mM as for the γ-Glu-Val generation activity. That is, if the activities are compared by using Vmax/Km as index of the activities, the ratio of γ-Glu-Val generation activity to the γ-Glu-Gly generation activity in the case of using Mn2+ as the cofactor can be calculated to be 0.20.
It is also known that GSHA derived from Proteus mirabilis, a kind of gram-negative bacteria, generates γ-glutamyl peptides by using Mg2+ or Mn2+ as a cofactor, as well as Glu, various kinds of amino acids, and ATP as substrates (Non-patent document 2). It has been reported that if the γ-Glu-Cys generation activity of GSHA derived from Proteus mirabilis is taken as 100%, the γ-Glu-Gly generation activity and γ-Glu-Val generation activity of the same correspond to 14.5% and 7.2%, respectively. That is, if the activities are compared on the basis of these relative activities, the ratio of γ-Glu-Val generation activity to the γ-Glu-Gly generation activity can be calculated to be 0.50.